Project Summary/Abstract Pancreatic cancer is the most deadly cancer type, with just an 8% 5-year survival. The most common subtype, pancreatic ductal adenocarcinoma (PDAC), is typified by the nearly universal KRAS mutational activation (~95%), followed by loss of the tumor suppressor CDKN2A (~95%), which encodes INK4A, an endogenous inhibitor of the CDK4/6 cell-cycle regulatory proteins. Loss of CDKN2A activity is a major step in KRAS- mediated PDAC tumorigenesis. KRAS and INK4A cooperate to accelerate PDAC metastatic development, in part, by converging to block RB tumor suppressor activity. While no anti-KRAS targeted therapies have reached the clinic, inhibitors of CDK4/6 are clinically approved. With the loss of CDKN2A being critical to allow KRAS-driven PDAC progression, restoration of CDKN2A function via pharmacologic inhibition of CDK4/6 should be an effective anti-KRAS therapeutic strategy. My preliminary results suggest that CDK4/6 inhibitors may benefit patient outcome in PDAC, especially in combination with ERK inhibitors, which block the most critical KRAS effector signaling pathway. Therefore, I propose to pre-clinically validate CDK4/6 inhibitors for anti-cancer activity, to discover synergistic combinations with other pharmacologic agents, and to identify potential resistance mechanisms which may limit their clinical effectiveness. In Specific Aim 1, I will validate CDK4/6 inhibitors alone and with ERK inhibitors for PDAC therapy by determining the short and long-term cellular consequences of inhibition in two-dimensional culture, three-dimensional organoid culture, and in vivo mouse models. To identify novel and clinically actionable small molecule inhibitors which sensitize resistant cell lines to CDK4/6 inhibitors, I will use high-throughput combinatorial compound screens in Aim 2 to identify compounds that synergistically enhance CDK4/6 inhibitor activity, and I will establish the mechanistic basis of synergy. Finally, in Aim 3, I will apply the Cancer Toolkit gain-of-function and a CRISPR/Cas9 loss-of-function genetic functional screens to identify novel mechanisms of resistance to CDK4/6 inhibition and will fully validate promising hits using pharmacologic and molecular biology techniques. These genetic screens will suggest small molecule combination therapy to overcome de novo or acquired CDK4/6 inhibitor resistance. In summary, my studies will make novel findings on CDK4/6 as cancer drivers and as therapeutic targets. To complete these studies, I will apply state-of-the-art methods that will provide me with training that will establish my expertise in all facets of anti-cancer drug discovery. Finally, my findings may be rapidly transitioned to clinical evaluation and will hopefully accelerate the development of new therapies for this deadly cancer.